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by the proteasome [1, 2]. The substrates contain linear motifs that
are recognized by a specialized E3, which ensures selectivity of the
process. These motifs are called degradation-inducing sequence
or shortly degron, if they are necessary and sufficient for recognition by the degradation machinery. Thus, modification of a stable
protein by a degron will lead to its destabilization. In rare cases,
proteins are also directly recognized by the proteasome and
degraded in a ubiquitin- independent way [3].
Due to the importance of proteolysis in eukaryotic cells, synthetic control of protein stability is a powerful tool to interfere
with regulatory mechanisms and to influence cellular activities. To
implement synthetic regulation, the activity of a degron is switched
from an inactive to an active state by a distinct signal. Until now,
signals like temperature, small molecules, nutrients, cell cycle stage
or light have been used to regulate the stability of conditional
degrons in eukaryotic cells [4–9]. To confer synthetic regulation
on a selected target protein, one of the degrons is fused to the target gene to create a cell line in which target protein abundance and
activity is controlled synthetically.
Development of such a degradation tool requires two protein
domains or sequences: a sensor domain that converts the signal
into an output, e.g., a conformational change and a degron that is
controlled by this switch. For generation of a light-controlled
degron, the light–oxygen–voltage (LOV) 2 photoreceptor domain
of Arabidopsis thaliana phototropin 1 was selected as sensor due to
precise knowledge about the molecular changes in the LOV2
domain after light-exposure. The LOV2 domain binds the cofactor
flavin-mononucleotide (FMN), which is excited by blue light.
Subsequently, the side-chain of a cysteine residue of LOV2 forms a
covalent bond with the C4a atom of FMN. This induces structural
changes in the LOV2 core resulting in unfolding of the so-called
Jα-helix at the carboxy terminus of the photoreceptor [10, 11].
The degradation sequence selected for the conditional degron
was derived from the well-studied murine ornithine decarboxylase
(ODC). The ODC degron sequences are located at the very
carboxy- terminus of the enzyme. It is somewhat unusual, as ubiquitin is not necessary for proteasomal degradation of ODC, whereas
the vast majority of proteasome substrates are degraded in an ubiquitin-dependent way [12]. The active ODC degron has two requirements: a cysteine-alanine motif located 19 amino acids upstream of
the C-terminus flanked by sequences without secondary structure
comprising a length of 37 amino acids in total [13, 14].
To generate the light-activated degron, the A. thaliana LOV2
domain was fused to a 23 amino acid long peptide of a synthetic
variant of the ODC degron called cODC1 [9, 15]. Several variants
of this construct were produced to assess the overall performance
of the degron, which was finally named photosensitive degron
(psd) module (Fig. 1a). The variants that were used in comparison
Christof Taxis
by the proteasome [1, 2]. The substrates contain linear motifs that
are recognized by a specialized E3, which ensures selectivity of the
process. These motifs are called degradation-inducing sequence
or shortly degron, if they are necessary and sufficient for recognition by the degradation machinery. Thus, modification of a stable
protein by a degron will lead to its destabilization. In rare cases,
proteins are also directly recognized by the proteasome and
degraded in a ubiquitin- independent way [3].
Due to the importance of proteolysis in eukaryotic cells, synthetic control of protein stability is a powerful tool to interfere
with regulatory mechanisms and to influence cellular activities. To
implement synthetic regulation, the activity of a degron is switched
from an inactive to an active state by a distinct signal. Until now,
signals like temperature, small molecules, nutrients, cell cycle stage
or light have been used to regulate the stability of conditional
degrons in eukaryotic cells [4–9]. To confer synthetic regulation
on a selected target protein, one of the degrons is fused to the target gene to create a cell line in which target protein abundance and
activity is controlled synthetically.
Development of such a degradation tool requires two protein
domains or sequences: a sensor domain that converts the signal
into an output, e.g., a conformational change and a degron that is
controlled by this switch. For generation of a light-controlled
degron, the light–oxygen–voltage (LOV) 2 photoreceptor domain
of Arabidopsis thaliana phototropin 1 was selected as sensor due to
precise knowledge about the molecular changes in the LOV2
domain after light-exposure. The LOV2 domain binds the cofactor
flavin-mononucleotide (FMN), which is excited by blue light.
Subsequently, the side-chain of a cysteine residue of LOV2 forms a
covalent bond with the C4a atom of FMN. This induces structural
changes in the LOV2 core resulting in unfolding of the so-called
Jα-helix at the carboxy terminus of the photoreceptor [10, 11].
The degradation sequence selected for the conditional degron
was derived from the well-studied murine ornithine decarboxylase
(ODC). The ODC degron sequences are located at the very
carboxy- terminus of the enzyme. It is somewhat unusual, as ubiquitin is not necessary for proteasomal degradation of ODC, whereas
the vast majority of proteasome substrates are degraded in an ubiquitin-dependent way [12]. The active ODC degron has two requirements: a cysteine-alanine motif located 19 amino acids upstream of
the C-terminus flanked by sequences without secondary structure
comprising a length of 37 amino acids in total [13, 14].
To generate the light-activated degron, the A. thaliana LOV2
domain was fused to a 23 amino acid long peptide of a synthetic
variant of the ODC degron called cODC1 [9, 15]. Several variants
of this construct were produced to assess the overall performance
of the degron, which was finally named photosensitive degron
(psd) module (Fig. 1a). The variants that were used in comparison
Christof Taxis